PROJECT SUMMARY Signaling metabolites are small molecules with routine functions in cellular energy metabolism that also act as signals to regulate diverse cellular pathways in response to a changing energy state. Signaling metabolites link nutrition to aging. Many of the emerging geroscience therapies that target mechanisms of aging have come from the discovery and understanding of signaling metabolites. The ketone body ?-hydroxybutyrate (BHB) is a new signaling metabolite. It is produced during fasting, dietary restriction, exercise, or carbohydrate restriction to keep the body's tissues supplied with energy when glucose is scarce. We now have growing evidence that it also functions as a signal, by inhibiting enzymes, binding directly to proteins as a post-translational modification, and activating receptors. Through its signaling activities, BHB regulates gene expression, inflammation, metabolism, senescence, and other cellular activities important to both aging and Alzheimer's disease (AD). We recently showed for the first time that ketogenic diet (KD), which stimulates endogenous production of BHB similar to fasting, improves survival in aging mice and prevents age-related declines in memory. We also found that KD improves memory in the hAPPJ20 AD mouse model, and reduces abnormal epileptiform discharges that contribute to memory decline. KD is a complex intervention, and though it is now being studied in clinical trials of AD, a better understanding of which aspects of KD are most helpful should lead to better targeted and more effective therapies. We have successfully developed an innovative toolset of dietary, chemical, and genetic tools to isolate the individual components of KD, including carbohydrate restriction, BHB, energy provision by BHB, and cellular signaling activities of BHB. We will use these tools to uncover the specific mechanisms by which BHB improves memory in normal aging and in AD mice (Aim 1), and reduces epileptiform discharges in AD mice (Aim 2). We will characterize key molecular changes that BHB causes in the proteomic landscape of the brain, including mapping the acetylome and new BHBylome (Aim 3). This project combines expertise in both geroscience and AD with a deep understanding of BHB biology to carry out closely aligned mechanistic studies using both aging and AD models. It examines the intersection of a molecular mechanism that is broadly relevant to aging (ketone bodies as metabolic signals) with one highly specific to AD (aberrant epilepsy-like network hypersynchrony). It is highly likely to stimulate further progress on AD because the mechanistic framework it generates will directly inform translational studies involving ketone body compounds and ketogenic diets. These data will help establish criteria for designing effective interventions, provide relevant intermediate biomarkers, and permit deeper investigation into the downstream molecular targets most relevant to AD.